This is a re-revised application. Oral tolerance refers to the long held observation that oral antigen results in systemic hypo-responsiveness. Oral antigen can suppress animal models of autoimmune diseases such as EAE and arthritis and is of benefit in models of stroke, atherosclerosis, and transplantation. Oral tolerance is mediated by multiple mechanisms including active suppression, anergy, and deletion. Nonetheless, a great deal remains to be learned about basic mechanisms of oral tolerance and gut associated immune responses. Our laboratory has a long-term interest in both basic mechanisms of oral tolerance and its use as immune therapy. In this competitive renewal we will address what we believe are critical questions related to understanding oral tolerance. We will use the C57B16 model of EAE induced by MOG and we will utilize an adoptive transfer system involving a newly described MOG TCR transgenic mouse which will allow us to measure the effect of oral tolerance on antigen specific T cell populations. We will address the following questions: l. What are the inductive events associated with oral tolerance and the generation of TGF-beta regulatory T cells in the gut? TGF-beta secreting Th3 cells and CD4+LAP+ cells are preferentially induced in the gut. Based on new preliminary data, we will investigate antigen presenting cells (B cells, macrophages, dendritic cells) in the mesenteric LN and Peyer's patches required for the induction of CD4+LAP+ T cells in the gut. We will employ a newly developed in vitro system to study isolated cell populations. 2. What is the role of ICOS in oral tolerance and in the generation of antigen-specific regulatory T cells induced by oral tolerance? ICOS is a new member of the CD28 family which is important in the activation and function of effector T cells. We have found upregulation of ICOS on T cells following oral antigen using RNA microarray analysis and by cell surface expression.. We have found a defect of oral tolerance in ICOS-/- mice which suggests it plays an important role in oral tolerance and the induction of regulatory T cells. 3. What are the mechanisms associated with enhancement of oral tolerance by orally administered adjuvants that polarize towards Th1 vs. Th2/3 responses. The gut preferentially generates Th2 and TGF-Beta (Th3) type responses. Orally active adjuvants may be crucial for the induction of immune responses via the gut of significant strength to be effective as immune therapy. We will investigate basic mechanisms of two unique adjuvants that we have found to be orally active and that polarize towards Thl vs. Th2/3 responses and their effect on the EAE model. The application has been re-revised in accordance with the reviewer's critiques with special attention to specific aim 1.